The physical integrity of soft and hard tissues is protected and maintained by salivary secretions over a wide range of conditions prevailing in the oral cavity. Histatins are a family of structurally related histidine-rich proteins present in parotid and submandibular/sublingual secretions of man and subhuman primates which exhibit a range of direct and indirect antimicrobial activities. These antimicrobial properties have been established by bioassays specifically designed to measure antifungal and antibacterial activities against oral candidal strains, cariogenic bacteria, and periodontal pathogens. of special interest is the fact that specific structural elements of histatins could be identified as functional domains in the mid-portion of the single polypeptide chain and that synthetic peptides representing such functional domains exhibit biological activities identical to or greater than those associated with the parent molecule. These features make histatins attractive candidates for therapeutic exploitation using either direct delivery systems or gene transfer approaches. While significant progress has been made in understanding the biology of histatins, several central questions remain to be answered. These questions pertain to the mechanisms of such multifunctional proteins, the boundaries of their antimicrobial spectrum, the conformational requirements for function, the role played by other salivary proteins after interaction with histatins, and how physiological parameters modulate biological activity. To gain insights into the mechanisms of candidacidal activity we will use genetic approaches to identify and characterize genes whose overexpression in multicopy plasmids confers resistance to histatins or renders histatin resistant mutants sensitive to killing by histatins. Building on our recent work showing protease treatment of candidal spheroplasts rendered them insensitive to histatins we plan to identify and characterize histatin receptor molecules on spheroplast membranes. The multifunctional nature of histatins will be addressed by identifying the functional domains responsible for growth inhibition of histatin-susceptible periodontal pathogens. Indirect antibacterial effects of histatins will be studied with enzyme kinetics by characterizing the inhibition of specific proteases and identify the mode of action of histatins on the neutralization of leukotoxin elaborated by the periodontal pathogen Actinobacillus actinomycetemcomitans. Recombinant methods will be employed to elucidate the effects induced by changing primary structures of histatins using functional domain replication and single amino acid substitutions. Since recent work with some histatins and synthetic histatin peptides with identical primary structures revealed functional and non-functional preparations we will use circular dichroism and nuclear magnetic resonance spectroscopy to determine the conformational requirements for function. Another goal is to study the interactions of histatins with other salivary proteins at the level of both complex formation and proteolysis in oral fluid using radiolabeled histatins and histatin peptides. Concentration measurement, functional analysis and several exocrine parameters will be determined in individual saliva samples. Correlation of these data will permit a realistic assessment of the role of histatins under physiological conditions.